Herbicide-resistant proteins, encoding genes, and uses thereof

ABSTRACT

Some embodiments of the present invention can include herbicide-resistant proteins, coding genes, and uses thereof. Certain herbicide-resistant proteins can comprises: (a) a protein consisting of an amino acid sequence shown in SEQ ID NO: 2; or (b) a protein consisting of an amino acid sequence at least 90% identical to that set forth in SEQ ID NO: 2. Other herbicide-resistant proteins of this disclosure can be suitable for expression in plants, can have resistance to herbicides (e.g., to phenoxy auxinherbicides), or both.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119(a)-(d) of ChinesePatent Application No. 201210570529.8 filed Dec. 25, 2012, entitled“Herbicide-resistant protein, encoding gene and use thereof” which isherein incorporated by reference in its entirety.

TECHNICAL FIELD Some aspects of the present application relate toherbicide-resistant proteins, encoding genes, and uses thereof, such asa 2,4-D-resistant protein, encoding gene and use thereof. BACKGROUND

Weeds can quickly run out of the valuable nutrients in the soil whichare necessary for the growth of crops and other target plants. Atpresent, there are many types of herbicides for weeds control, amongwhich is a particularly popular herbicide, glyphosate.Glyphosate-resistant crops have been developed, such as corn, soybean,cotton, beet, wheat, and rice, and the like. Thus, it is possible tospray glyphosate in the fields planted with glyphosate-resistant cropsto control weeds without significant damage to the crops.

Glyphosate has been widely used all over the world for more than 20years, resulting in the overdependence on the technology of glyphosateand glyphosate-tolerant crops. In addition, high selection pressure hasbeen forced to the naturally more glyphosate-tolerant plants among thewild weed species or the plants which have developed resistance toglyphosate activity. It has reported that a few weeds have developedresistance to glyphosate, including broad-leaved weeds and gramineousweeds, such as Swiss ryegrass, Lolium multiflorum, Eleusine indica,Ambrosia artemisiifolia, Conyza canadensis, Conyza bonariensis andPlantago lanceolata. In addition, the weeds which are not theagricultural problem before the widespread use of glyphosate-tolerantcrops also gradually prevailed, and are difficult to be controlled withglyphosate-tolerant crops. These weeds mainly exist along with (but notonly with) broad-leaved weeds which are difficult to be controlled, suchas species from Amaranthus, Chenopodium, Taraxacum and Commelinaceae.

In the area of glyphosate-resistant weeds or the weed species which aredifficult to be controlled, growers can make up the weakness of theglyphosate through tank-mixing or using other herbicide which cancontrol the omissive weeds. In most cases, a popular and effectivetank-mixing partner used to control broad-leaved weeds is2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D has been used to controlbroad-spectrum broad-leaved weeds more than 65 years under agricultureand non-crop conditions, and is still one of the most widely usedherbicides in the world. The limit for further use of 2,4-D is that itsselectivity in dicotyledonous plants (such as soybeans or cotton) isvery low. Therefore, 2,4-D is generally not used on (and generally notclose to) sensitive dicotyledonous plants. In addition, the use of 2,4-Don gramineous crops is limited to a certain extent by the properties ofthe potential crop damage. The combination of 2,4-D and glyphosate hasalready been used to provide a stronger sterilization process beforeplanting the no-till soybeans and cotton. However, due to thesensitivity of these dicotyledonous species to 2,4-D, thesesterilization processes must be carried out 14 to 30 days beforeplanting.

Same as MCPA, 2-methyl-4-chloropropionic acid and 2,4-D propionic acid,2,4-D is also a phenoxy alkanoic acid herbicide. 2,4-D is used toselectively control broad-leaved weeds in many monocotyledonous cropssuch as corn, wheat and rice, without serious damage to the targetcrops. 2,4-D is a synthetic auxin derivative of which the function is todisorder the normal cytohormone homeostasis and to hinder the balance ofcontrolled growth.

2,4-D shows different levels of selectivity on certain plants (forexample, dicotyledonous plants are more sensitive than gramineousplants). Different 2,4-D metabolisms in different plants are oneexplanation for the different levels of selectivity. Plants usuallymetabolize 2,4-D slowly. Thus, different activities of targeted pointsare more likely to explain different responses to 2,4-D of plants. Plantmetabolism of 2,4-D is usually achieved through two steps of metabolism,i.e. the conjugation with amino acids or glucose following thehydroxylation in general.

As time goes on, the microbial populations have gradually developedeffective, alternative pathways to degrade this particular foreignsubstance, which result in the complete mineralization of 2,4-D.Continuous application of herbicides on microbes can be used to selectthe microorganisms which use herbicides as carbon sources so as to makea competitive advantage in the soil. For this reason, 2,4-D wascurrently formulated with a relatively short soil half-life period andwithout obvious legacy effect on the subsequent crops, which promotesthe application of 2,4-D herbicide.

Ralstonia eutropha is one organism of which the ability for degrading2,4-D has been widely studied. The gene encoding the enzyme in the firstenzymatic step of mineralization pathway is tfdA. TfdA catalyzes theconversion of 2,4-D acid into dichlorophenol (DCP) throughα-oxoglutarate-dependent dioxygenase reaction. DCP hardly has herbicideactivity compared with 2,4-D. TfdA is used to introduce 2,4-D resistanceinto dicotyledonous plants which are usually sensitive to 2,4-D (such ascotton and tobacco) in transgenic plants.

A number of tfdA type genes have been identified which encode proteinscapable of degrading 2,4-D in the environment. Many homologs are similarwith tfdA (amino acid identity >85%) and have similar enzyme activitywith tfdA. However, a large number of homologs have significantly loweridentity (25-50%) with tfdA while contain characteristic residuesassociated with α-oxoglutarate-dependent dioxygenase Fe²⁺ dioxygenases.Therefore, the substrate specificities of these different dioxygenasesare indefinite. A unique instance which has low homology (28% amino acididentity) with tfdA is rdpA from Sphingobium herbicidovorans. It hasbeen shown that this enzyme catalyzes the first step in themineralization of (R)-2,4-D propionic acid (and other (R)-phenoxypropionic acids) and 2,4-D (phenoxyacetic acid).

With the emergence of glyphosate-resistant weeds and the expandedapplication of 2,4-D herbicide, some embodiments include theintroduction of 2,4-D resistance into the target plants sensitive to2,4-D. At present, no reports have been found about the expressionlevels of herbicide-resistant protein 24DT02 in plants and theirherbicide tolerance.

SUMMARY

In some instances, the purpose of the present application is to provideherbicide-resistant proteins, coding genes, and uses thereof. Thepresent application is, in some instances, intended to provide a 24DT02gene which has higher herbicide tolerance in plants.

In one aspect, the present application provides a herbicide-resistantprotein, comprising:

-   (a) a protein consisting of an amino acid sequence shown in SEQ ID    NO: 2; or-   (b) a protein with the activity of aryloxy alkanoate di-oxygenase    which is derived from the amino acid sequence in (a) by replacing    and/or deleting and/or adding one or several amino acids in the    same; or-   (c) a protein consisting of an amino acid sequence at least 90%    identical to that set forth in SEQ ID NO: 2.

In some embodiments, said herbicide-resistant protein is a proteinconsisting of an amino acid sequence at least 95% identical to that setforth in SEQ ID NO: 2.

In some embodiments, said herbicide-resistant protein is a proteinconsisting of an amino acid sequence at least 99% identical to that setforth in SEQ ID NO: 2.

In one aspect, the present application provides a herbicide-resistantgene, comprising:

-   (a) a nucleotide sequence encoding said herbicide-resistant protein;    or-   (b) a nucleotide sequence capable of hybridizing with the nucleotide    sequence as defined in (a) under stringent conditions and encoding a    protein with the aryloxy alkanoate di-oxygenase activity; or-   (c) the nucleotide sequence set forth in SEQ ID NO: 1.    The stringent conditions might be as follows: hybridization in 6×SSC    (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution at    65° C. and followed by washing membrane one time using 2×SSC, 0.1%    SDS and 1×SSC, 0.1% SDS, respectively.

In another aspect, the present application also provides an expressioncassette, comprising said herbicide-resistant gene under the regulationof operably linked regulatory sequence.

In one aspect, the present application further provides a recombinantvector, comprising said herbicide-resistant gene or said expressioncassette.

In another aspect, the present application further provides a transgenichost cell comprising said herbicide-resistant gene or the expressioncassette, wherein said transgenic host cell comprises plant cells,animal cells, bacteria, yeast, baculovirus, nematodes, or algae. In someembodiments, the transgenic host might be selected from a groupconsisting of plant, animal, bacteria, yeast, baculovirus, nematodes,and algae.

In some embodiments, said transgenic host is a plant selected from thegroup consisting of soybean, cotton, corn, rice, wheat, beet andsugarcane.

In one aspect, the present application further provides a method forproducing a herbicide-resistant protein, comprising steps of:

-   -   obtaining the transgenic host cells;    -   cultivating the transgenic host cells under the conditions        allowing for the production of the herbicide-resistant protein;        and    -   recovering the herbicide-resistant protein.

In one aspect, the present application also provides a method forextending the target range of herbicides, comprising a step ofco-expressing of the nucleotide encoding said herbicide-resistantprotein or the expression cassette with at least a second nucleotideencoding a herbicide-resistant protein different from theherbicide-resistant protein as described above or saidherbicide-resistant protein encoded by the expression cassette.

In some embodiments, said second nucleotide encodes glyphosate-resistantprotein, glufosinate ammonium-resistant protein, 4-hydroxyphenylpyruvatedioxygenase, acetohydroxyacid synthase, cytochrome protein orprotoporphyrinogen oxidase.

Alternatively, said second nucleotide is a dsRNA which inhibitsimportant genes in target insect pest.

In yet another aspect, the present application provides a transgenichost cell co-expressing the nucleotide encoding said herbicide-resistantprotein or the expression cassette with at least a second nucleotideencoding a herbicide-resistant protein different from theherbicide-resistant protein as described above or saidherbicide-resistant protein encoded by the expression cassette.

In some embodiments, said second nucleotide encodes glyphosate-resistantprotein, glufosinate ammonium-resistant protein, 4-hydroxyphenylpyruvatedioxygenase, acetohydroxyacid synthase, cytochrome protein orprotoporphyrinogen oxidase.

Alternatively, said second nucleotide is a dsRNA which inhibitsimportant genes in target insect pest.

In some embodiments of the present application, the herbicide-resistantprotein 24DT02 is expressed in a transgenic plant accompanied by theexpressions of one or more glyphosate-resistant proteins and/orglufosinate-ammonium-resistant proteins. Such a co-expression of morethan one kind of herbicide-resistant protein in a same transgenic plantcan be achieved by transfecting and expressing the genes of interest inplants through genetic engineering. In addition, herbicide-resistantprotein 24DT02 can be expressed in one plant (Parent 1) through geneticengineering operations and glyphosate-resistant protein and/orglufosinate-ammonium-resistant protein can be expressed in a secondplant (Parent 2) through genetic engineering operations. The progenyplants expressing all genes of Parent 1 and Parent 2 can be obtained bycrossing Parent 1 and Parent 2.

RNA interference (RNAi) refers to a highly conserved and effectivedegradation phenomenon of specific homologous mRNA induced bydouble-stranded RNA (dsRNA) during evolution. Therefore, RNAi technologycould be applied to specifically knock out or shut down the expressionof a specific gene.

In yet another aspect, the present application also provides a methodfor selecting transformed plant cells, comprising the steps oftransforming multiple plant cells with the herbicide-resistant gene orthe expression cassette and cultivating said cells at a herbicideconcentration which allows the growth of the transformed cellsexpressing the herbicide-resistant gene or the expression cassette whilekilling the un-transformed cells or inhibiting the growth of theun-transformed cells, wherein the herbicide is a phenoxy auxin.

In one aspect, the present application also provides a method forcontrolling weeds, comprising a step of applying an effective amount ofherbicides to the field planted with crops containing saidherbicide-resistant gene, said expression cassette or said recombinantvector.

In some embodiments, the herbicide is a phenoxy auxin.

In another aspect, the present application also provides a method forprotecting plants from the damage caused by herbicides, comprising thestep of introducing said herbicide-resistant gene, said expressioncassette or said recombinant vector into plants such that the obtainedplants produce a certain quantity of herbicide-resistant proteinsufficient to protect them from the damage caused by herbicides.

In some embodiments, the said herbicide is a phenoxy auxin or aryloxyphenoxy propionate and said plants are selected from the groupconsisting of soybean, cotton, corn, rice, wheat, beet and sugarcane.

In one aspect, the present application also provides a method forcontrolling glyphosate-resistant weeds in a field planted withglyphosate-tolerant plants, comprising a step of applying an effectiveamount of herbicides to the field planted with glyphosate-tolerantplants containing said herbicide-resistant gene, said expressioncassette or said recombinant vector.

In some embodiments, said herbicide is a phenoxy auxin and saidglyphosate-tolerant plant is monocotyledon or dicotyledon.

In another aspect, the present application also provides a method forconferring crops with resistance to 2,4-D herbicides, comprising thesteps of introducing said herbicide-resistant gene, said expressioncassette or said recombinant vector into plants.

In some embodiments, said plants are selected from the group consistingof soybean, cotton, corn, rice, wheat, beet and sugarcane.

In yet another aspect, the present application relates to a method forcontrolling weeds comprising a step of applying an effective amount ofherbicides to the field planted with crops containing theherbicide-resistant gene of present application.

In some embodiments, the herbicide-resistant gene is produced from atransgenic host cell selected from the group consisting of plant cells,animal cells, bacteria, yeast, baculovirus, nematodes and algae.

In some embodiments, the plant is selected from the group consisting ofsoybean, cotton, corn, rice, wheat, beet and sugarcane.

In some embodiments, the nucleotide encoding the herbicide-resistantprotein or the herbicide-resistant gene is co-expressed in the plantwith at least a second nucleotide encoding a herbicide-resistant proteindifferent from that of present application.

In some embodiments, said second nucleotide encodes glyphosate-resistantprotein, glufosinate ammonium resistant protein, 4-hydroxyphenylpyruvatedioxygenase, acetolactate synthase, cytochromes protein orprotoporphyrinogen oxidase.

In some embodiments, the herbicide is a phenoxy auxin.

The herbicide-resistant gene, said expression cassette or saidrecombinant vector is introduced into plants. The conventional methodsused in present application to introduce foreign DNA into plant cellsinclude but are not limited to Agrobacterium-mediated transfection,Particle Bombardment, direct intake of DNA into protoplast,electroporation or silicon-mediated DNA introduction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the scheme to construct the recombinant cloning vectorDBN01-T containing 24DT02 nucleotide sequence;

FIG. 2 shows the scheme to construct the recombinant expression vectorDBN100223 containing 24DT02 nucleotide sequence;

FIG. 3 shows the scheme to construct the recombinant expression vectorDBN100223N containing control sequence;

FIG. 4 shows the herbicide-resistant effect of the transgenicArabidopsis T₁ plant;

FIG. 5 shows the scheme to construct the recombinant expression vectorDBN100212 containing 24DT02 nucleotide sequence;

FIG. 6 shows the scheme to construct the recombinant expression vectorDBN100212N containing control sequence.

DETAILED DESCRIPTION

The 2,4-D resistance genes and subsequent resistance crops according topresent application provide a good choice to controlglyphosate-resistance (or high tolerance or succession) broad-leavedweed species in crops. 2,4-D is a broad-spectrum, relatively cheap andpowerful broad-leaved herbicides. If stronger crop tolerance in bothdicotyledons and monocots could be provided, good efficacies could beprovided for growers. 2,4-D-tolerant transgenic dicotylenons also have ahigher flexibility in application time and administration amount.Another use of the 2,4-D herbicide-tolerance trait is that it could beused to prevent damage to normal sensitive crops such as 2,4-D drift,volatilization, transformation (or other remote movement phenomenon),misuse, destruction and the like. Various mixtures of different phenoxyauxins have been widely used to treat specific weed spectrum andenvironmental conditions in different areas. Using 24DT02 gene in plantscan provide protections against broader-spectrum phenoxy auxin herbicideso as to improve the flexibility and controllable weed spectrum andprovide protections to the full range of commercially available phenoxyauxin drift or other long distance phenoxy herbicides damages.

Phenoxy auxin herbicides are usually formulated as active acids, butsome commercialized preparations are formulated as one of severalcorresponding ester preparations. Since general plant esterases inplants can convert these esters into active acids, they are alsoconsidered to be the substrates of 24DT02 enzyme in plants. Similarly,they can also be the organic or inorganic salts of the correspondingacids. When expressing chiral propionic acid, propionic acid salt orpropionic ester herbicides, even if different CAS numbers may correspondto an optically pure compound. When denominating the herbicides, westill consider that racemic (R, S) or optically pure (R or S) enantiomeris a same herbicide. The possible dosage ranges can be those treatedalone or combined with other herbicides in the applications in crops ornon-crops.

It has been identified that the 24DT02 gene possesses thecharacteristics to allow the application of phenoxy auxin herbicide inplants after expressing the genetically engineered 24DT02 in plants, ofwhich the inherent tolerance does not exist or is not enough to allowthe application of these herbicides. In addition, 24DT02 gene canprovide protection on phenoxy auxin herbicides when the naturaltolerance is not enough to allow selectivity in plants. One, two orseveral phenoxy auxin herbicides can be continuously or tank-mixedlycombined with it to treat plants only comprising 24DT02 gene. Dosagerange of each phenoxy auxin herbicide used to control the broad-spectrumof dicotyledonous weeds ranges from 25 to 4000 g ae/ha, more generallyfrom 100 to 2000 g ae/ha. Combination of these herbicides belonging todifferent chemical classes and having different action modes in a samefield (continuously or tank-mixedly) can control most potential weedswhich are intentioned to be controlled by the herbicides.

Glyphosate is widely used because it controls very broad spectrum ofbroad-leaved and gramineous weed species. However, the repeated use ofglyphosate in the application of glyphosate-tolerant crops and non-cropshas (and will continue to) selectively resulted in the succession of theweeds to species with more natural tolerance or glyphosate-resistantbiotype. Most of the herbicide resistance management strategiesrecommend using effective amount of tank-mixed herbicide partners as away to delay the appearance of resistant weeds. The herbicide partnersprovide the control of a same species but with different modes ofaction. The overlay of 24DT02 gene and glyphosate-tolerance trait(and/or other herbicide-tolerance traits) can provide the control ofglyphosate-resistant weed species (broad-leaved weed species controlledby one or more phenoxy auxins) in glyphosate-tolerance crops byselectively applying glyphosate and phenoxy auxin (such as 2,4-D) on thesame crops. Applications of these herbicides might be the individual useof single herbicide composition in a tank mixture containing two or moreherbicides with different action models simultaneously or sequentially(e.g. before planting, before seedling emergence or after seedlingemergence) (interval time ranged from 2 hours to 3 months).Alternatively, compositions of any number of herbicides representingevery class of compound could be used at any time (from within 7 monthsafter planting to the time of harvest (or, as to a single herbicide, itrefers to preharvest interval in which the shortest one is selected)).

Flexibility is very important in the control of broad-leaved weeds,e.g., application time, dosage of a single herbicide and the ability tocontrol stubborn or resistant weeds. The dosage of glyphosate whichoverlays with glyphosate-resistant gene/24DT02 gene can range from 250to 2500 g ae/ha; the dose of (one or more) phenoxy auxin herbicides canrange from 25 to 4000 g ae/ha. The optimum combination of theapplication time can depend on the specific conditions, species and theenvironment.

Herbicide formulations (such as esters, acids or salt formulas orsoluble concentrates, emulsified concentrates or soluble solutions) andadditives of tank-mixture (such as adjuvant or compatilizer) can affectthe weed control of a given herbicide or a combination of one or morekinds of herbicides. Any chemical combinations of any of aboveherbicides are comprised in the scope of this application.

The benefits of the combination of two or more action modes in improvingthe controlled weed spectrum and/or natural species with more toleranceor resistance weed species can be extended to chemicals capable ofproducing other herbicide tolerances besides glyphosate-tolerance incrops through artificial means (transgenic or non-transgenic). In fact,the following resistance characteristics can be encoded alone or bemultiply overlayed so as to provide the ability to effectively controlor prevent weeds from succession to any category of the above-mentionedherbicide resistances: glyphosate resistance (such as resistant plant orbacteria, EPSPS, GOX, GAT), glufosinate-ammonium resistance (such asPAT, Bar), acetolactate synthase (ALS) inhibitory herbicide resistance(such as imidazolidinone, sulfonylurea, triazole pyrimidine,sulphonanilide, pyrimidine thiobenzoate and other chemical resistancegenes such as AHAS, Csrl, SurA etc.), bromoxynil resistance (such asBxn), resistance to inhibitor of HPPD (4-Hydroxyphenylpyruvatedioxygenase), resistance to inhibitor of phytoene desaturase (PDS),resistance to photosystem II inhibitory herbicide (such as psbA),resistance to photosystem I inhibitory herbicide, resistance toprotoporphyrinogen oxidase IX (PPO) inhibitory herbicide (such asPPO-1), phenylurea herbicide resistance (such as CYP76B1), dicambadegrading enzyme etc.

As to other herbicides, some of other ALS inhibitors includetriazolopyrimidine benzenesulfonamide (cloransulam-methyl, diclosulam,flumetsulam, metosulam and pyrimidino triazoles sulfonamide), pyrimidinethiobenzoate and flucarbazone. Some HPPD inhibitors include mesotrione,isoxaflutole and sulcotrione. Some PPO inhibitors include flumioxazin,butafenacil, carfentrazone, sulfentrazone and diphenyl oxide (such asacifluorfen, fomesafen, Lactofen and oxyfluorfen).

In addition, 24DT02 genes can be overlayed alone with one or more otherinput (such as insect resistance, fungus resistance or stress tolerance)or output (such as the increased yield, improved oil mass, improvedfiber quality) traits, or overlayed with one or more other input (suchas insect resistance, fungus resistance or stress tolerance) or output(such as the increased yield, improved oil mass, improved fiber quality)traits after overlaying with other herbicide-resistant cropcharacteristics. Therefore, this application can provide the ability toflexibly and economically control any number of agronomy pests and acomplete agronomy solution to improve crop quality.

24DT02 gene in this application can degrade 2,4-D, which is the basis ofimportant herbicide-resistant crops and of the possibility of selectionmarkers.

Almost all the herbicide combinations for broad-leaved weeds could becontrolled by the transgenic expression of 24DT02 gene. 24DT02 gene asan excellent herbicide tolerant crop trait can be overlayed with, forexample, other herbicide-tolerant crop characteristics, such asglyphosate resistance, glufosinate-ammonium resistance, ALS inhibitor(such as imidazolidinone, sulfonylurea and triazolopyrimidinebenzenesulfonamides) resistance, bromoxynil resistance, HPPD inhibitorresistance, PPO inhibitor resistance and the like) and insect resistancetraits (Cry1Ab, Cry1F, Vip3, other bacillus thuringiensis protein orinsect-resistant protein derived from the non-bacillus). In addition,24DT02 gene can be used as a selection marker to assist the selection ofthe primary transformant of plants genetically modified with anothergene or genogroup.

Phenoxy alkanoate group can be used to introduce stable acid functionalgroups into herbicides. Acidic groups can import phloem activity by“acid capture” (the property required by herbicide effect) so as to beintegrated into the new herbicides for activity purpose. There are manycommercially available and experimental herbicides as substrates of24DT02. Therefore, tolerances to other herbicides can be obtained byusing present application.

The crop herbicide-tolerance trait of this application can be used in anew combination with other crop herbicide-tolerance traits (includingbut not limited to glyphosate tolerance). Because of the newly acquiredresistance or inherent tolerance to herbicides (such as glyphosate), thecombinations of these traits produce new methods to control weedspecies. Therefore, in addition to crop herbicide-tolerance traits,present application also includes new methods for controlling weeds byusing herbicides, in which the said herbicide-tolerance is obtainedthrough the enzyme produced by the transgenic crops.

The present application can be applied to a variety of plants, such asarabidopsis, tobacco, soybean, cotton, rice, corn and brassica. Thepresent application can also be applied to a variety of othermonocotyledonous (such as gramineous herbage or grassy carpet) anddicotyledonous crops (such as alfalfa, clover and tree species, etc.).Similarly, 2,4-D (or other 24DT02 substrates) can be applied moreactively to gramineous crops with moderate tolerance, and the resultedtolerance of which traits are raised will provide growers thepossibility to use these herbicides with more effective dosage andbroader administration time without the risk of crop injury.

The genomes of plants, plant tissues or plant cells described in thisapplication refer to any genetic materials in the plants, plant tissuesor plant cells, and include the nucleus, plasmids and mitochondrialgenomes.

The “resistance” described herein is heritable, and allows the plants togrow and reproduce under the case that effective treatment is applied tothe given plants using common herbicide. Even if a certain damage of theplant caused by herbicides occurs, the plant can still be considered“resistance”. The term “tolerance” described herein is broader than theterm “resistance” and includes “resistance” and the improved ability ofparticular plant resistant to the various degree of damages induced bythe herbicides which result generally in the damages of the wild typeplants with the same genotypes under the same herbicide dosage.

As described herein, polynucleotides and/or nucleotides form a complete“gene” and encode proteins or polypeptides in the host cells ofinterest. The polynucleotides and/or nucleotides in the presentapplication can be under the control of the regulatory sequences of thetarget host.

DNA exists typically as double strands. In such an arrangement, onestrand is complementary with the other, and vice versa. When DNA isreplicated in plants, other complementary strands of DNA are alsogenerated. Therefore, the polynucleotides exemplified in the sequencelisting and complementary strands thereof are comprised in thisapplication. The “coding strand” generally used in the art refers to astrand binding with an antisense strand. To express a protein in vivo,one strand of the DNA is typically transcribed into a complementarystrand of a mRNA, which serves as the template of protein expression. Infact, a mRNA is transcribed from the “antisense” strand of DNA. “Sensestrand” or “coding strand” contains a series of codons (codon is atriplet of nucleotides that codes for a specific amino acid), whichmight be read as open reading frames (ORF) to generate target proteinsor peptides. RNA and PNA (peptide nucleic acid) which are functionallyequivalent with the exemplified DNA were also contemplated in thisapplication.

Nucleic acid molecule or fragments thereof were hybridized with theherbicide-resistant gene under stringency condition in this application.Any regular methods of nucleic acid hybridization or amplification canbe used to identify the existence of the herbicide-resistant gene inpresent application. Nucleic acid molecules or fragments thereof arecapable of specifically hybridizing with other nucleic acid moleculesunder certain conditions. In present application, if two nucleic acidmolecules can form an antiparallel nucleic acid structure with doublestrands, it can be determined that these two molecules can hybridizewith each other specifically. If two nucleic acid molecules arecompletely complementary, one of two molecules is called as the“complement” of the other one. In this application, when everynucleotide of a nucleic acid molecule is complementary with thecorresponding nucleotide of another nucleic acid molecule, it isidentified the two molecules are “completely complementary”. If twonucleic acid molecules can hybridize with each other so that they cananneal to and bind to each other with enough stability under at leastnormal “low-stringency” conditions, these two nucleic acids areidentified as “minimum complementary”. Similarly, if two nucleic acidmolecules can hybridize with each other so that they can anneal to andbind to each other with enough stability under normal “high-stringency”conditions, it is identified that these two nucleic acids are“complementary”. Deviation from “completely complementary” can beallowed, as long as the deviation does not completely prevent the twomolecules to form a double-strand structure. A nucleic acid moleculewhich can be taken as a primer or a probe must have sufficientlycomplementary sequences to form a stable double-strand structure in thespecific solvent at a specific salt concentration.

In this application, basically homologous sequence refers to a nucleicacid molecule, which can specifically hybridize with the complementarystrand of another matched nucleic acid molecule under “high-stringency”condition. The stringency conditions for DNA hybridization can be anysuitable conditions, such as treatment with 6.0× sodium chloride/sodiumcitrate (SSC) solution at about 45° C. and washing with 2.0×SSC at 50°C. For example, the salt concentration in the washing step is selectedfrom 2.0×SSC and 50° C. for the “low-stringency” conditions and 0.2×SSCand 50° C. for the “high-stringency” conditions. In addition, thetemperature in the washing step ranges from 22° C. for the“low-stringency” conditions to 65° C. for the “high-stringency”conditions. Both temperature and the salt concentration can varytogether or only one of these two variables varies. In some embodiments,the stringency condition used in this application might be as below. SEQID NO:1 is specifically hybridized in 6.0×SSC and 0.5% SDS solution at65° C. Then the membrane was washed one time in 2×SSC and 0.1% SDSsolution and 1×SSC and 0.1% SDS solution, respectively.

Therefore, the herbicide-resistant sequences which can hybridize withSEQ ID NO: 1 under stringency conditions were comprised in thisapplication. These sequences were at least about 40%-50% homologous orabout 60%, 65% or 70% homologous, even at least 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homologous to thesequences of present application.

The present application provides functional proteins. “Functionalactivity” (or “activity”) as described herein means the activity ofproteins/enzymes (alone or combined with other protein) in thisapplication to degrade herbicide or reduce the herbicide activity. Theplants which produce the proteins of this application can produce suchan effective amount of proteins that, when treating plants withherbicides, the protein expression level is enough to provide the plantswith complete or partial resistance or tolerance to herbicides (generaldosage if there are no specific instructions). Herbicides are usuallyapplied at the dosage capable of killing the target plants, normaldosage and concentration applied in the field. In some embodiment, plantcells and plants of this application are protected from the growthinhibition or damage caused by herbicide treatment. The transformedplants and plant cells of the present application can have resistance ortolerance to 2,4-D herbicides, which means that the transformed plantsand plant cells can survive in the condition with effective amount of2,4-D herbicides.

Genes and proteins described in the present application include not onlythe specifically exemplified sequences, but also parts and/or fragments(including deletion(s) in and/or at the end of the full-length protein),variants, mutants, substitutes (proteins containing substituted aminoacid(s)), chimeras and fusion proteins retaining the herbicide-resistantactivity thereof. The said “variants” or “variation” refers to thenucleotide sequences encoding the same one protein or encoding anequivalent protein having herbicide-resistant activity. The said“equivalent protein” refers to the proteins that have the same or thesubstantially same bioactivity of herbicide-resistant activity as thatof the claimed proteins.

The “fragment” or “truncation” of the DNA or protein sequences asdescribed in this application refers to a part or an artificiallymodified form thereof (e.g., sequences suitable for plant expression) ofthe original DNA or protein sequences (nucleotides or amino acids)involved in present application. The sequence length of said sequence isvariable, but it is long enough to ensure that the (encoded) protein isherbicide-resistant protein. In some cases (especially expression inplants), it is advantageous to use a truncated gene which encodes atruncated protein. The truncated gene can encode 40, 41, 42,43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% of the whole protein.

Due to redundancy of the genetic codons, a variety of different DNAsequences can encode one same amino acid sequence. Substituted DNAsequences encoding one same or substantially same protein can also beachieved. These different DNA sequences are encompassed by thisapplication. The said “substantially same” sequence refers to a sequencein which certain amino acids are substituted, deleted, added orinserted, but herbicide-resistant activity thereof is not substantiallyaffected, and also includes the fragments remaining theherbicide-resistant activity.

Substitution, deletion or addition of some amino acids in amino acidsequences in this application can be achieved by any suitable technique.In some embodiment, such an amino acid change includes: minorcharacteristics change, e.g., substitution of reserved amino acids whichdo not significantly influence the folding and/or activity of theprotein; short deletion, usually a deletion of about 1-30 amino acids;short elongation of amino or carboxyl terminal, such as a methionineresidue elongation at amino terminal; short connecting peptide, such asabout 20-25 residues in length.

The examples of conservative substitution are the substitutionshappening in the following amino acids groups: basic amino acids (suchas arginine, lysine and histidine), acidic amino acids (such as glutamicacid and aspartic acid), polar amino acids (e.g., glutamine andasparagine), hydrophobic amino acids (such as leucine, isoleucine, andvaline), aromatic amino acids (e.g., phenylalanine, tryptophan andtyrosine), and small molecular amino acids (such as glycine, alanine,serine and threonine and methionine). Some amino acid substitutionsgenerally not changing specific activity have been described in, forexample, Protein edited by N. Neurath and R. L. Hill, published byAcademic Press, New York in 1979. Some substitutions are Ala/Ser,Val/Ile, Asp/Glu, Thu/Ser, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, andreverse substitutions thereof.

Such a substitution may happen outside of the regions which areimportant to the molecular function and still cause the production ofactive polypeptides. For the polypeptide of the present application, theamino acid residues which are required for their activity and chosen asthe unsubstituted residues can be identified according to the knownmethods of the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (see, e.g. Cunningham and Wells, 1989,Science 244:1081-1085). The latter technique is carried out byintroducing mutations in every positively charged residue in themolecule and detecting the herbicide-resistant activity of the obtainedmutation molecules, so as to identify the amino acid residues which areimportant to the activity of the molecules. Enzyme-substratesinteraction sites can also be determined by analyzing itsthree-dimensional structure, which can be determined through sometechniques such as nuclear magnetic resonance (NMR) analysis,crystallography, or photoaffinity labeling (see, for example, de Vos etal., 1992, Science 255:306-312; Smith, et al., 1992, J. Mol. Biol224:899-904; Wlodaver, et al., 1992, FEBS Letters 309:59-64).

Therefore, amino acid sequences which have certain homology with theamino acid sequences set forth in SEQ ID No. 2 are also comprised inthis application. The sequence similarity/homology between thesesequences and the sequences described in the present application can bemore than 60%, more than 75%, more than 80%, more than 90% or more than95%. The polynucleotides and proteins in the present application can, insome instances, be defined according to more specific ranges of thehomology and/or similarity. For example, they have a homology and/orsimilarity of 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with thesequences described in this application.

Regulatory sequences described in this application include but are notlimited to a promoter, transit peptide, terminator, enhancer, leadingsequence, introns and other regulatory sequences that can be operablylinked to the said 24DT02 gene.

The said promoter is a promoter expressible in plants, wherein said “apromoter expressible in plants” refers to a promoter which ensures thatthe coding sequences bound with the promoter can be expressed in plantcells. The promoter expressible in plants can be a constitutivepromoter. The examples of promoters capable of directing theconstitutive expression in plants include but are not limited to 35Spromoter derived from Cauliflower mosaic virus, ubi promoter, promoterof GOS2 gene derived from rice and the like. Alternatively, the promoterexpressible in plants can be a tissue-specific promoter, which meansthat the expression level directed by this promoter in some planttissues such as in chlorenchyma, is higher than that in other tissues ofthe plant (can be measured through the conventional RNA test), such asthe PEP carboxylase promoter. Alternatively, the promoter expressible inplants can be wound-inducible promoters as well. Wound-induciblepromoters or promoters that direct wound-inducible expression mannersrefer to the promoters by which the expression level of the codingsequences can be increased remarkably compared with those under thenormal growth conditions when the plants are subjected to mechanicalwound or wound caused by the gnaw of insects. The examples ofwound-inducible promoters include but are not limited to the promotersof genes of protease inhibitor of potatoes and tomatoes (pin I and pinII) and the promoters of maize protease inhibitor gene (MPI).

The said transit peptide (also called paracrine signal sequence orleader sequence) directs the transgenosis products into specificorganelles or cellular compartments. For the receptor protein, the saidtransit peptide can be heterogeneous. For example, sequences encodingchloroplast transit peptide are used to lead to chloroplast; or ‘KDEL’reserved sequence is used to lead to the endoplasmic reticulum or CTPPof the barley lectin gene is used to lead to the vacuole.

The said leader sequences include but are not limited to small RNA virusleader sequences, such as EMCV leader sequence (encephalomyocarditisvirus 5′ non coding region); Potato virus Y leader sequences, such asMDMV (Maize dwarf mosaic virus) leader sequence; human immunoglobulinheavy chain binding protein (BiP); untranslated leader sequence of thecoat protein mRNA of Alfalfa Mosaic virus (AMV RNA4); Tobacco Mosaicvirus (TMV) leader sequence.

The said enhancer includes but is not limited to Cauliflower Mosaicvirus (CaMV) enhancer, Figwort Mosaic virus (FMV) enhancer, CarnationsEtched Ring virus (CERV) enhancer, Cassava Vein Mosaic virus (CsVMV)enhancer, Mirabilis Mosaic virus (MMV) enhancer, Cestrum yellow leafcurling virus (CmYLCV) enhancer, Cotton leaf curl Multan virus (CLCuMV),Commelina yellow mottle virus (CoYMV) and peanut chlorotic streak mosaicvirus (PCLSV) enhancer.

For the application of monocotyledon, the said introns include but arelimited to maize hsp70 introns, maize ubiquitin introns, Adh intron 1,sucrose synthase introns or rice Act1 introns. For the application ofdicotyledonous plants, the said introns include but are not limited toCAT-1 introns, pKANNIBAL introns, PIV2 introns and “super ubiquitin”introns.

The said terminators can be the proper polyadenylation signal sequencesplaying a role in plants. They include but are not limited topolyadenylation signal sequence derived from Agrobacterium tumefaciensnopaline synthetase (NOS) gene, polyadenylation signal sequence derivedfrom protease inhibitor II (pin II) gene, polyadenylation signalsequence derived from peas ssRUBISCO E9 gene and polyadenylation signalsequence derived from α-tubulin gene.

The term “operably linked” described in this application refers to thelinking of nucleic acid sequences, which provides the sequences therequired function of the linked sequences. The term “operably linked”described in this application can be the linkage of the promoter withthe sequences of interest, which makes the transcription of thesesequences under the control and regulation of the promoter. When thesequence of interest encodes a protein and the expression of thisprotein is required, the term “operably linked” indicates that thelinking of the promoter and said sequence makes the obtained transcriptto be effectively translated. If the linking of the promoter and thecoding sequence results in transcription fusion and the expression ofthe encoding protein are required, such a linking is generated to makesure that the first translation initiation codon of the obtainedtranscript is the initiation codon of the coding sequence.Alternatively, if the linking of the promoter and the coding sequenceresults in translation fusion and the expression of the encoding proteinis required, such a linking is generated to make sure that the firsttranslation initiation codon of the 5′ untranslated sequence is linkedwith the promoter, and such a linking way makes the relationship betweenthe obtained translation products and the open reading frame encodingthe protein of interest meet the reading frame. Nucleic acid sequencesthat can be “operably linked” include but are not limited to sequencesproviding the function of gene expression (e.g., gene expressionelements, such as a promoter, 5′ untranslated region, introns,protein-coding region, 3′ untranslated region, polyadenylation sitesand/or transcription terminators); sequences providing the function ofDNA transfer and/or integration (e.g., T-DNA boundary sequences,recognition sites of site-specific recombinant enzyme, integraserecognition sites); sequences providing selectable function (e.g.,antibiotic resistance markers, biosynthetic genes); sequences providingthe function of scoring markers; sequences assistant with the operationof sequences in vitro or in vivo (polylinker sequences, site-specificrecombinant sequences) and sequences providing replication function(e.g., origins of replication of bacteria, autonomously replicatingsequences, centromeric sequences).

This application can confer new herbicide resistant trait(s) to theplants while adverse effects on phenotypes including yield are notobserved. The plants of present application can tolerate against 2×, 3×,4× or 5× general application level of at least one subjected herbicide.The improvement of these resistance levels is in the scope of presentapplication. For example, it is possible to foreseeably optimize andfurther develop many kinds of known technologies in the art so as toincrease the expression of a given gene.

In some aspects of the present application, said herbicide-resistantprotein is 24DT02 amino acid sequence as shown in SEQ ID NO: 2 of thesequence listing. Said herbicide-resistant gene can be 24DT02 nucleotidesequence as shown in SEQ ID NO: 1 of the sequence listing. In order tobe applied to plants, said herbicide-resistant gene also contains,besides coding region of the protein encoded by 24DT02 nucleotidesequence, other elements, such as encoding regions which encode transitpeptides, the coding regions which encode selective marker proteins orthe proteins which confer resistance to insect.

The herbicide-resistant protein 24DT02 as describe herein can betolerant to most phenoxy auxin herbicides. The genomes of the plants inpresent application contain exogenous DNAs which contain 24DT02nucleotide sequence. The plants are protected from the threat ofherbicides by expressing effective amount of this protein. “Effectiveamount” refers to the amount which causes no damage or causes slightdamage to the plant; in some instances, the plants are morphologicallynormal or almost morphologically normal, and could be cultivated underthe common means for the consumption and/or generation of products.

The expression level of herbicide-resistance crystal proteins (ICP) inthe plant materials can be determined using various methods described inthis field, such as the method of quantifying mRNA encoding theherbicide-resistant protein in the tissue through using specificprimers, or the method of quantifying the herbicide-resistant proteindirectly and specifically.

Some embodiments of the present application provide herbicide-resistantproteins, coding genes, and uses thereof that can have one or more offollowing advantages:

1. Strong herbicide-resistance activity. Herbicide-resistant protein24DT02 can be strongly resistant to herbicides, such as to phenoxy auxinherbicides, particularly 2,4-D.

2. Broad herbicide-resistance spectrum. The herbicide-resistant protein24DT02 can show high resistance to a variety of plant phenoxy auxinherbicides, therefore it has broad application prospect on the plants.

The technical solutions of this application will be further describedthrough the appended figures and examples as disclosed herein.

EXAMPLES

The technical solutions of herbicide-resistant proteins, coding genesand uses thereof in present application will be further illustratedthrough the following examples.

Example 1 The Obtaining and Synthesis of 24DT02 Gene Sequence

1. Obtaining of 24DT02 Gene Sequence

Amino acid sequence of 24DT02 herbicide-resistant protein (298 aminoacids) was shown as SEQ ID NO: 2 in the sequence listing; the nucleotidesequence (897 nucleotides) encoding the corresponding amino acidsequence of 24DT02 herbicide-resistant protein (298 amino acids) wasshown as SEQ ID NO: 1 in the sequence listing.

2. Synthesis of the Nucleotide Sequence as Described above

The 24DT02 nucleotide sequence (shown as SEQ ID NO: 1 in the sequencelisting) was synthesized by GenScript CO., LTD, Nanjing, P.R. China. Thesynthesized 24DT02 nucleotide sequence (SEQ ID NO: 1) was linked with aSpeI restriction site at the 5′ end and a KasI restriction site at the3′ end.

At the same time, the substituted 24DT02 nucleotide sequence (shown asSEQ ID NO: 3 in the sequence listing) was also synthesized, in which theMet²⁶⁷ was substituted with Leu. The synthesized, substituted 24DT02nucleotide sequence (SEQ ID NO: 3) was linked with a SpeI restrictionsite at the 5′ end and a KasI restriction site at the 3′ end.

At the same time, the truncated 24DT02 nucleotide sequence (shown as SEQID NO: 4 in the sequence listing) was also synthesized, which iscomposed of the amino acids from 1 to 295 of 24DT02 amino acid sequence.The synthesized, truncated 24DT02 nucleotide sequence (SEQ ID NO: 4) waslinked with a SpeI restriction site at the 5′ end and a KasI restrictionsite at the 3′ end.

At the same time, the added 24DT02 nucleotide sequence (shown as SEQ IDNO: 5 in the sequence listing) was also synthesized, in which threeamino acids Ala, Leu and Val were added after the 298th amino acid of24DT02 amino acid sequence. The synthesized, added 24DT02 nucleotidesequence (SEQ ID NO: 5) was linked with a SpeI restriction site at the5′ end and a KasI restriction site at the 3′ end.

Example 2 Construction of Arabidopsis Recombinant Expression Vectors andthe Transfection of Agrobacterium with the Recombinant ExpressionVectors

1. Construction of the Arabidopsis Recombinant Cloning Vector DBN01-TContaining 24DT02 Nucleotide Sequence

The synthesized 24DT02 nucleotide sequence was sub-cloned into cloningvector pGEM-T (Promega, Madison, USA, CAT: A3600), to get recombinantcloning vector DBN01-T following the instructions of Promega pGEM-Tvector, and the construction process was shown in FIG. 1 (wherein theAmp is ampicillin resistance gene; f1 is the replication origin of phagef1; LacZ is initiation codon of LacZ; SP6 is the promoter of SP6 RNApolymerase; T7 is the promoter of T7 RNA polymerase; 24DT02 is 24DT02nucleotide sequence (SEQ ID NO: 1); MCS is multiple cloning sites).

The recombinant cloning vector DBN01-T was then transformed into E. coliT1 competent cell (Transgen, Beijing, China, the CAT: CD501) throughheat shock method. The heat shock conditions were as follows: 50 μl ofE. coli T1 competent cell and 10 μl of plasmid DNA (recombinant cloningvector DBN01-T) were incubated in water bath at 42° C. for 30 seconds.Then the E. coli cells were incubated in water bath at 37° C. for 1 h(100 rpm in a shaking incubator) and then were grown on a LB plate (10g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L Agar and pH wasadjusted to 7.5 with NaOH) coated on the surface with IPTG (Isopropylthio-beta-D-galactose glucoside), X-gal(5-bromine-4-chlorine-3-indole-beta-D-galactose glucoside) andampicillin (100 mg/L) overnight. The white colonies were picked out andcultivated in LB broth (10 g/L Tryptone, 5 g/L yeast extract, 10 g/LNaCl, 100 mg/L ampicillin and pH was adjusted to 7.5 with NaOH) at 37°C. overnight. The plasmids thereof were extracted using alkaline lysismethod as follows: the bacterial liquid was centrifuged for 1 min at12000 rpm, the supernatant was discarded and the pellet was resuspendedin 100 μl of ice-chilled solution I (25 mM Tris-HCl, 10 mM EDTA(ethylenediaminetetraacetic acid) and 50 mM glucose, pH=8.0); then 150μl of freshly prepared solution II (0.2 M NaOH, 1% SDS (sodium dodecylsulfate)) was added and the tube was reversed 4 times, mixed and thenput on ice for 3-5 minutes; 150 μl of cold solution III (4 M potassiumacetate and 2 M acetic acid) was added, thoroughly mixed immediately andincubated on ice for 5-10 minutes; the mixture was centrifuged at 12000rpm at 4° C. for 5 minutes, two volumes of anhydrous ethanol were addedinto the supernatant, mixed and then placed at room temperature for 5minutes; the mixture was centrifuged at 12000 rpm at 4° C. for 5minutes, the supernatant was discarded and the pellet was dried afterwashed with 70% ethanol (V/V); 30 μl TE (10 mM Tris-HCl, 1 mM EDTA,pH=8.0) containing RNase (20 μg/ml) was added to dissolve theprecipitate; the mixture was incubated at 37° C. in a water bath for 30min to digest RNA and stored at −20° C. for the future use.

After the extracted plasmids were confirmed with restriction enzymesSpeI and KasI, the positive clones were verified through sequencing. Theresults showed that said 24DT02 nucleotide sequence inserted into therecombinant cloning vector DBN01-T was the sequence set forth in SEQ IDNO: 1 in the sequence listing, indicating that 24DT02 nucleotidesequence was correctly inserted.

The synthesized, substituted 24DT02 nucleotide sequence was insertedinto cloning vector pGEM-T to get recombinant cloning vector DBN02-Tfollowing the process for constructing recombinant cloning vectorDBN01-T as described above, wherein mi24DT02 was substituted 24DT02nucleotide sequence (SEQ ID NO: 3). The substituted 24DT02 nucleotidesequence in the recombinant cloning vector DBN02-T was verified to becorrectly inserted with restriction enzyme digestion and sequencing.

The synthesized, truncated 24DT02 nucleotide sequence was inserted intocloning vector pGEM-T to get recombinant cloning vector DBN03-Tfollowing the process for constructing cloning vector DBN01-T asdescribed above, wherein mt24DT02 was truncated 24DT02 nucleotidesequence (SEQ ID NO: 4). The truncated 24DT02 nucleotide sequence in therecombinant cloning vector DBN03-T was verified to be correctly insertedwith restriction enzyme digestion and sequencing.

The synthesized, added 24DT02 nucleotide sequence was inserted intocloning vector pGEM-T to get recombinant cloning vector DBN04-Tfollowing the process for constructing cloning vector DBN01-T asdescribed above, wherein ma24DT02 was added 24DT02 nucleotide sequence(SEQ ID NO: 5). The added 24DT02 nucleotide sequence in the recombinantcloning vector DBN04-T was verified to be correctly inserted withrestriction enzyme digestion and sequencing.

2. Construction of the Arabidopsis Recombinant Expression VectorDBN100223 Containing 24DT02 Nucleotide Sequence

The recombinant cloning vector DBN01-T and expression vector DBNBC-01(Vector backbone: pCAMBIA2301, available from CAMBIA institution) weredigested with restriction enzymes SpeI and KasI. The cleaved 24DT02nucleotide sequence fragment was ligated between the restriction sitesSpeI and KasI of the expression vector DBNBC-01 to construct therecombinant expression vector DBN100223. The construction scheme wasshown in FIG. 2 (Spec: spectinomycin gene; RB: right border; AtUbi10:Arabidopsis Ubiquitin (Ubiquitin) 10 gene promoter (SEQ ID NO: 6);24DT02: 24DT02 nucleotide sequence (SEQ ID NO: 1); Nos: terminator ofnopaline synthetase gene (SEQ ID NO: 7); prCaMV35S: Cauliflower mosaicvirus 35S promoter (SEQ ID NO:8); PAT: glufosinate acetyl transferasegene (SEQ ID NO:9); tCaMV35S: Cauliflower mosaic virus 35S terminater(SEQ ID NO: 10); LB: left border).

The recombinant expression vector DBN100223 was transformed into E. coliT1 competent cells with heat shock method as follows: 50 μl of E. coliT1 competent cell and 10 μl of plasmid DNA (recombinant expressionvector DBN100223) were incubated in water bath at 42° C. for 30 seconds.Then the E. coli cells were incubated in water bath at 37° C. for 1 hour(100 rpm in a shaking incubator) and then were grown on a LB solid plate(10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L Agar and pHwas adjusted to 7.5 with NaOH) containing 50 mg/L spectinomycin at 37°C. for 12 hours. The white colonies were picked out and cultivated in LBbroth (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 50 mg/Lspectinomycin and pH was adjusted to 7.5 with NaOH) at 37° C. overnight.The plasmids thereof were extracted using alkaline lysis method. Afterthe extracted plasmids were confirmed with restriction enzymes SpeI andKasI, the positive clones were verified through sequencing. The resultsshowed that the nucleotide sequence between restriction sites SpeI andKasI in the recombinant expression vector DBN100223 was the nucleotidesequence set forth in SEQ ID NO: 1 in the sequence listing, e.g., 24DT02nucleotide sequence.

Following the process for constructing recombinant expression vectorDBN100223 as described above, recombinant cloning vector DBN02-T wasdigested with restriction enzymes SpeI and KasI to cleave thesubstituted 24DT02 nucleotide sequence which then was inserted into theexpression vector DBNBC-01 to get the recombinant expression vectorDBN100223-i. Restriction enzyme digestion and sequencing verified thatthe nucleotide sequence between restriction sites SpeI and KasI in therecombinant expression vector DBN100223-i was the substituted 24DT02nucleotide sequence.

Following the process for constructing recombinant expression vectorDBN100223 as described above, recombinant cloning vector DBN03-T wasdigested with restriction enzymes SpeI and KasI to cleave the truncated24DT02 nucleotide sequence which then was inserted into the expressionvector DBNBC-01 to get the recombinant expression vector DBN100223-t.Restriction enzyme digestion and sequencing verified that the nucleotidesequence between restriction sites SpeI and KasI in the recombinantexpression vector DBN100223-t was the truncated 24DT02 nucleotidesequence.

Following the process for constructing recombinant expression vectorDBN100223 as described above, recombinant cloning vector DBN04-T wasdigested with restriction enzymes SpeI and KasI to cleave the added24DT02 nucleotide sequence which then was inserted into the expressionvector DBNBC-01 to get the recombinant expression vector DBN100223-a.Restriction enzyme digestion and sequencing verified that the nucleotidesequence between restriction sites SpeI and KasI in the recombinantexpression vector DBN100223-a was the added 24DT02 nucleotide sequence.

3. Construction of the Arabidopsis Recombinant Expression VectorDBN100223N Containing Control Sequence

Following the process for constructing recombinant cloning vectorDBN01-T comprising 24DT02 nucleotide sequence as described in part 1 ofExample 2, recombinant cloning vector DBN01R-T containing controlsequence was constructed by using control sequence (SEQ ID NO: 11). Thepositive clones were verified through sequencing. The results showedthat the natural nucleotide sequence inserted into the recombinantcloning vector DBN01R-T was the sequence set forth in SEQ ID NO: 11 inthe sequence listing, indicating that control nucleotide sequence wascorrectly inserted.

Following the process for constructing recombinant expression vectorDBN100223 containing 24DT02 nucleotide sequence as described in part 2of Example 2, recombinant expression vector DBN100223N containingnatural sequence was constructed using the natural sequence and theconstruction process was shown in FIG. 3 ((Vector backbone: pCAMBIA2301,available from CAMBIA institution); Spec: spectinomycin gene; RB: rightborder; AtUbi10: Arabidopsis Ubiquitin (Ubiquitin) 10 gene promoter (SEQID NO: 6); mN: control sequence (SEQ ID NO: 11); Nos, terminator ofnopaline synthetase gene (SEQ ID NO: 7); prCaMV35S: Cauliflower mosaicvirus 35S promoter (SEQ ID NO:8); PAT: glufosynat acetyl transferasegene (SEQ ID NO:9); tCaMV35S: Cauliflower mosaic virus 35S terminator(SEQ ID NO: 10); LB: left border). The positive clones were verifiedthrough sequencing. The results showed that the control sequenceinserted into the recombinant expression vector DBN100223N was thesequence set forth in SEQ ID NO: 11 in the sequence listing, indicatingthat control sequence was correctly inserted.

4. Transfection of Agrobacterium tumefaciens with the ArabidopsisRecombinant Expression Vectors

The correctly constructed recombinant expression vectors DBN100223,DBN100223-i, DBN100223-t, DBN100223-a and DBN100223N (control sequence)were transfected into Agrobacterium GV3101 following liquid nitrogenrapid-freezing method as follows: 100 μL Agrobacterium GV3101 and 3 μLplasmid DNA (recombinant expression vector) were put into liquidnitrogen for 10 minutes and then incubated in water bath at 37° C. for10 minutes. Then the transfected Agrobacterium GV3101 cells wereinoculated in LB broth and cultivated at 28° C., 200 rpm for 2 hours andspreaded on a LB plate containing 50 mg/L of rifampicin (Rifampicin) and100 mg/L of spectinomycin until positive mono colonies appeared. Thepositive mono colonies were picked up and cultivated and the plasmidsthereof were extracted. Recombinant expression vectors DBN100223,DBN100223-i, DBN100223-t, and DBN100223-a DBN100223N (control sequence)were verified with restriction enzymes StyI and BglII and recombinantexpression vector DBN100223N (control sequence) was verified withrestriction enzymes Styl and BglI. The results showed that therecombinant expression vectors DBN100223, DBN100223-i, DBN100223-t,DBN100223-a and DBN100223N (natural sequence) were correct in structure,respectively.

Example 3 Obtaining of the Arabidopsis Plant with Inserted 24DT02Nucleotide Sequence

The wild-type Arabidopsis seeds were suspended in 0.1% agarose solutionand kept at 4° C. for 2 days so as to meet the need for dormancy toensure the synchronous germination of seeds. Vermiculite and horses dungwere mixed together and irrigated wet with water underground. The soilmixture was dewatered for 24 hours. The pretreated seeds were cultivatedin the soil mixture and covered with a moisturizing mask for 7 days. Theseeds were germinated and the plants were cultivated in a greenhouse ata constant temperature of 22° C. with constant moisture of 40-50% and along day condition with the light intensity of 120-150 μmol/m²s (16hours of light/8 hours of darkness). The plants were irrigated withHoagland nutrient solution at first and then with deionized water tokeep the soil moist but not drenched.

Floral dip method was used to transform Arabidopsis. One or more YEPmedia containing 100 mg/L of spectinomycin and 10 mg/L of rifampicin of15-30 ml were inoculated with the selected Agrobacterium colonies as apreculture. The preculture was incubated at 28° C. and 220 rpmovernight. Each preculture was used to inoculate two cultures of 500 mlYEP media containing spectinomycin (100 mg/L) and rifampicin (10 mg/L)and the cultures were incubated at 28° C. in a shaking incubatorovernight. Cultures were centrifuged at 8700×g for 10 minutes at roomtemperature to precipate cells and the obtained supernatant wasdiscarded. The cell pellets were gently resuspended in 500 ml ofpermeable medium which contains ½×MS salts/vitamin B5, 10% (w/v)sucrose, 0.044 μM Benzylaminopurine (10 μl/L (1 mg/ml stock solution inDMSO)) and 300 μl/L Silvet L-77. About 1 month old plants were soaked inthe medium for 15 seconds and the latest inflorescences were ensured tobe submerged. Then plants were put down by side and covered (transparentor non-transparent) for 24 hours, then washed with water and placedvertically. The plants were cultivated at 22° C. in a light cycle of 16hours of light/8 hours of darkness. Seeds were harvested after beingsoaked for 4 weeks.

The newly harvested T₁ seeds (24DT02 nucleotide sequence, 24DT02substituted nucleotide sequence, 24DT02 truncated nucleotide sequences,24DT02 added nucleotide sequence and natural sequence) were dried atroom temperature for 7 days. The seeds were cultivated in germinationplates (26.5×51 cm), 200 mg T₁ seeds (about 10000 seeds)/plate. Theseeds had been suspended in 40 ml of 0.1% agarose solution and stored at4° C. for 2 days to meet the need for dormancy to ensure the synchronousgermination of seeds.

Vermiculite and horses dung were mixed together and irrigated wet withwater underground and drained through gravity. The pretreated seeds (40ml each one) were uniformly planted on the soil mixture by using pipetteand covered with moisturizing mask for 4 to 5 days. The mask was removed1 day before the initial transformant selection by sprayingglufosinate-ammonium (selection of the co-transformed PAT gene) aftergermination.

On 7 days after planting (DAP) and 11 DAP respectively, the T1 plants(cotyledon stage and 2-4 leaves stage, respectively) were sprayed with0.2% of Liberty herbicide solution (200 g ai/L glufosinate-ammonium)using DeVilbiss compressed air nozzle at a spraying volume of 10 ml/disc(703 L/ha) so as to provide effective amount of glufosinate-ammonium(280 g ai/ha) for each application. The survival plants (activelygrowing plants) were verified 4 to 7 days after the last spraying andtransferred into the square pot (7 cm×7 cm) made from vermiculite andhorses dung (3-5 plants per pot). The transplanted plants were coveredwith moisturizing mask for 3-4 days and placed in culture room at 22° C.or directly into the greenhouse as described above. Then the mask wasremoved and the plants were planted in greenhouse (22±5° C., 50±30% RH,14 hours of lighting: 10 hours of darkness, minimum 500 g/m²s¹ naturallight+complement light) at least one day before testing the ability of24DT02 to provide the resistance to phenoxy auxin herbicide.

Example 4 Herbicide Resistance Effect Test of the Transgenic Arabidopsis

24DT02 gene was used to transform Arabidopsis for the first time. Atfirst, T₁ transformants were selected from the background ofun-transformed seeds, using glufosinate-ammonium selection scheme. About40000 T₁ seeds were screened among which 195 strains of T1 generationpositive transformants (PAT gene) were identified, i.e. thetransformation efficiency was about 0.5%. Herbicide resistance effecttests to 2,4-D dimethyl ammonium salt and agroxone of Arabidopsis T₁plants transformed with 24DT02 nucleotide sequence, substituted 24DT02nucleotide sequence, truncated 24DT02 nucleotide sequence, added 24DT02nucleotide sequence, control nucleotide sequence respectively andwild-type Arabidopsis plants were performed after 18 days of planting.

Arabidopsis T₁ plants transformed with 24DT02 nucleotide sequence,substituted 24DT02 nucleotide sequence, truncated 24DT02 nucleotidesequence, added 24DT02 nucleotide sequence and control nucleotidesequence respectively and wild-type Arabidopsis plants were sprayed with2,4-D dimethyl ammonium salt (560 g ae/ha, 1-fold concentration infield), agroxone (560 g ae/ha, 1-fold concentration in field) and blanksolvent (water). Resistance conditions of the plants were counted 7 daysand 14 days after spraying. Plants with growth conditions consistentwith blank solvent (water) 7 days after spaying were classified ashighly resistant plants; Plants with curly rosette leaves 7 days afterspaying were classified as moderately resistant plants; Plants incapableof bolting 14 days after spaying were classified as low-resistant plantsand the dead plants 14 days after spaying were classified asnon-resistant plants. Because each Arabidopsis T₁ plant was anindependent transformation event, significant differences of individualT₁ responses were expected under a given dose. The results were shown inTable 1 and FIG. 4.

TABLE 1 Herbicide resistance results of transgenic Arabidopsis T₁ plantsHighly Moder- Arabidopsis resis- ately Lowly Non- Treatment genotypetant resistant resistant resistant Sum Blank 24DT02 21 0 0 0 21 solvent24DT02-i 18 0 0 0 18 (H₂O) 24DT02-t 19 0 0 0 19 24DT02-a 17 0 0 0 17Control 20 0 0 0 20 Wild 31 0 0 0 31 560 g ae/ha 24DT02 9 4 0 5 18 2,4-D24DT02-i 8 4 1 4 17 dimethyl 24DT02-t 7 5 0 4 16 ammonium 24DT02-a 10 32 3 18 (1x 2,4-D) Control 0 0 0 18 18 Wild 0 0 0 20 20 560 g ae/ha24DT02 8 6 2 4 18 agroxone 24DT02-i 9 5 1 6 21 (1xMCPA) 24DT02-t 8 4 2 418 24DT02-a 8 5 1 3 17 Control 0 0 0 17 17 Wild 0 0 0 16 16

For Arabidopsis, 50 g ae/ha of 2,4-D and agroxone was the effective doseto distinguish the sensitive plants from plants with average resistance.Results shown in Table 1 and FIG. 4 indicated that the 24DT02 geneconferred herbicide resistance to individual Arabidopsis plants (onlyparts of the plants have the resistance because insertion sites of T1generation plants are random. Therefore the resistance gene expressionlevels were different, resulting in the different levels of resistance),especially the phenoxy auxin herbicides. The wild-type Arabidopsisplants and Arabidopsis plants transformed with control sequence had noresistance to phenoxy auxin herbicide. In addition, resistance levels to2,4-D dimethyl ammonium salt and agroxone of Arabidopsis T₁ plantstransformed with 24DT02 nucleotide sequence, substituted 24DT02nucleotide sequence, truncated 24DT02 nucleotide sequence and added24DT02 nucleotide sequence didn't show significant differences.

Example 5 Construction of the Corn Recombinant Expression Vector andTransfection of Agrobacterium with Recombinant Expression Vector

1. Construction of the Corn Recombinant Expression Vector DBN100212Containing 24DT02 Nucleotide Sequence

The recombinant cloning vector DBN01-T and expression vector DBNBC-02(Vector backbone: pCAMBIA2301, available from CAMBIA institution) weredigested with restriction enzymes SpeI and KasI. The cleaved 24DT02nucleotide sequence fragment was ligated between the restriction sitesSpeI and KasI of the expression vector DBNBC-01 to construct therecombinant expression vector DBN100212. SpeI and KasI restriction sitesin the expression vector DBNBC-01 were also introduced usingconventional enzyme digestion method. The construction scheme was shownin FIG. 5 (Spec: spectinomycin gene; RB: right border; Ubi: maizeUbiquitin (Ubiquitin) 1 gene promoter (SEQ ID NO: 12); 24DT02: 24DT02nucleotide sequence (SEQ ID NO: 1); Nos: terminator of nopalinesynthetase gene (SEQ ID NO: 7); PMI: phosphomannose isomerase gene (SEQID NO: 13); LB: left border).

The recombinant expression vector DBN100212 was transformed into E. coliT1 competent cells with heat shock methods as follows: 50 μl of E. coliT1 competent cell and 10 μl of plasmid DNA (recombinant expressionvector DBN100212) were incubated in water bath at 42° C. for 30 seconds.Then the E. coli cells were incubated in water bath at 37° C. for 1 hour(100 rpm in a shaking incubator) and then were grown on a LB solid plate(10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L Agar and pHwas adjusted to 7.5 with NaOH) containing 50 mg/L spectinomycin(Spectinomycin) at 37° C. for 12 hours. The white colonies were pickedout and cultivated in LB broth (10 g/L Tryptone, 5 g/L yeast extract, 10g/L NaCl, 50 mg/L spectinomycin and pH was adjusted to 7.5 with NaOH) at37° C. overnight. The plasmids thereof were extracted using alkalinelysis method. After the extracted plasmids were confirmed withrestriction enzymes SpeI and KasI, the positive clones were verifiedthrough sequencing. The results showed that the nucleotide sequencebetween restriction sites SpeI and KasI in the recombinant expressionvector DBN100212 was the nucleotide sequence set forth in SEQ ID NO: 1in the sequence listing, i.e. 24DT02 nucleotide sequence.

Following the process for constructing recombinant expression vectorDBN100212 as described above, recombinant cloning vector DBN02-T weredigested with restriction enzymes SpeI and KasI to cleave thesubstituted 24DT02 nucleotide sequence which then was inserted into theexpression vector DBNBC-01 to get the recombinant expression vectorDBN100212-i. Restriction enzyme digestion and sequencing verified thatthe nucleotide sequence between restriction sites SpeI and KasI in therecombinant expression vector DBN100212-i was the substituted 24DT02nucleotide sequence.

Following the process for constructing recombinant expression vectorDBN100212 as described above, recombinant cloning vector DBN03-T weredigested with restriction enzymes SpeI and KasI to cleave the truncated24DT02 nucleotide sequence which then was inserted into the expressionvector DBNBC-01 to get the recombinant expression vector DBN100212-t.Restriction enzyme digestion and sequencing verified that the nucleotidesequence between restriction sites SpeI and KasI in the recombinantexpression vector DBN100212-t was the truncated 24DT02 nucleotidesequence.

Following the process for constructing recombinant expression vectorDBN100212 as described above, recombinant cloning vector DBN04-T weredigested with restriction enzymes SpeI and KasI to cleave the added24DT02 nucleotide sequence which then was inserted into the expressionvector DBNBC-01 to get the recombinant expression vector DBN100212-a.Restriction enzyme digestion and sequencing verified that the nucleotidesequence between restriction sites SpeI and KasI in the recombinantexpression vector DBN100212-a was the added 24DT02 nucleotide sequence.

2. Construction of the Corn Recombinant Expression Vector DBN100212NContaining Control Nucleotide Sequence

Following the process for constructing recombinant cloning vectorDBN01-T containing 24DT02 nucleotide sequences described in part 1 ofExample 2, recombinant cloning vector DBN01R-T containing controlsequence was constructed by using control sequence (SEQ ID NO: 11). Thepositive clones were verified through sequencing. The results showedthat the natural nucleotide sequence inserted into the recombinantcloning vector DBN01R-T was the sequence set forth in SEQ ID NO: 11 inthe sequence listing, indicating that control nucleotide sequence wascorrectly inserted.

Following the process for constructing recombinant expression vectorDBN100212 containing 24DT02 nucleotide sequence as described in part 1of Example 5, recombinant expression vector DBN100212N containingnatural sequence was constructed by using the natural sequence and theconstruction process was shown in FIG. 6 (Vector backbone: pCAMBIA2301,available from CAMBIA institution); Spec: spectinomycin gene; RB: rightborder; ZmUbi1: maize Ubiquitin (ubiquitin) 1 gene promoter (SEQ ID NO:12); mN: control sequence (SEQ ID NO: 11); Nos: terminator of nopalinesynthetase gene (SEQ ID NO: 7); PMI: phosphomannose-isomerase gene (SEQID NO: 13); LB: left border). The positive clones were verified throughsequencing. The results showed that the control sequence inserted intothe recombinant expression vector DBN100223N was the sequence set forthin SEQ ID NO: 11 in the sequence listing, indicating that the controlnucleotide sequence was correctly inserted.

3. Transfection of Agrobacterium tumefaciens with Corn RecombinantExpression Vectors

The correctly constructed recombinant expression vectors DBN100212,DBN100212-i, DBN100212-t, DBN100212-a and DBN100212N (control sequence)were transfected into Agrobacterium LBA4404 (Invitrogen, Chicago, USA,Cat. No: 18313-015) following liquid nitrogen rapid-freezing method asfollows: 100 μL Agrobacterium LBA4404 and 3 μL plasmid DNA (recombinantexpression vector) were put into liquid nitrogen and kept for 10 minutesand then incubated in water bath at 37° C. for 10 minutes. Then thetransfected Agrobacterium LBA4404 cells were inoculated in LB tube andcultivated at 28° C., 200 rpm for 2 hours and spreaded on a LB platecontaining 50 mg/L of rifampicin (Rifampicin) and 100 mg/L ofspectinomycin until positive mono colonies appeared. The positive monocolonies were picked up and cultivated and the plasmids thereof wereextracted. Recombinant expression vectors DBN100212, DBN100212-i,DBN100212-t and DBN100212-a were verified with restriction enzymes EcoRIand BglII and DBN100212N (control sequence) was verified withrestriction enzymes StyI and BglI. The results showed that therecombinant expression vectors DBN100212, DBN100212-i, DBN100212-t,DBN100212-a and DBN100212N (natural sequence) were correct instructures, respectively.

Example 6 Obtaining and Verification of the Transgenic Corn Plants withInserted 24DT02 Nucleotide Sequence

According to the conventional Agrobacterium transfection method, themaize cultivar Zong 31 (Z31) was cultivated in sterilized conditions andthe young embryo was co-cultivated with the Agrobacterium strainsconstructed in part 3 of Example 5 so as to introduce T-DNAs in therecombinant expression vectors DBN100212, DBN100212-i, DBN100212-t,DBN100212-a and DBN100212N (natural sequence) constructed in part 1 and2 of Example 5 (including corn Ubiquitin 1 gene promoter sequence,24DT02 nucleotide sequence, 24DT02 substituted nucleotide sequence,24DT02 truncated nucleotide sequence, 24DT02 added nucleotide sequence,control nucleotide sequence, PMI gene and Nos terminator sequence) intothe maize genome. Maize plants containing 24DT02 nucleotide sequence,24DT02 substituted nucleotide sequence, 24DT02 truncated nucleotidesequence, 24DT02 added nucleotide sequence and control nucleotidesequence respectively were obtained and at the same time wild type cornplant was taken as a control.

As to the Agrobacterium-mediated transfection of maize, in brief,immature maize young embryo was isolated from corns and contacted withAgrobacterium suspension, in which the Agrobacterium can deliver the24DT02 nucleotide sequence into at least one cell of one young embryo.(Step 1: infection step). In this step, optionally, young embryo wasimmersed in Agrobacterium suspension (OD₆₆₀=0.4-0.6, infection medium(4.3 g/L of MS salt, MS vitamins, 300 mg/L of casein, 68.5 g/L ofsucrose, 36 g/L of glucose, 40 mg/L of Acetosyringone (AS), 1 mg/L of2,4-dichlorophenoxyacetic acid (2,4-D), pH=5.3)) to initiate theinoculation. Young embryo and Agrobacterium were cocultivated for aperiod (3 days) (Step 2: cocultivation step). In some embodiments, theYoung embryo was cultivated on a solid medium (4.3 g/L of MS salt, MSvitamins, 300 mg/L of casein, 20 g/L of sucrose, 10 g/L of glucose, 100mg/L of Acetosyringone (AS), 1 mg/L of 2,4-dichlorophenoxyacetic acid(2,4-D) and 8 g/L of Agar, pH=5.8) after the infection step. After thiscocultivation step, a selective “recovery” step could be preceded. Inthe “recovery” step, the recovery medium (4.3 g/L of MS salt, MSvitamins, 300 mg/L of casein, 30 g/L of sucrose, 1 mg/L of2,4-dichlorophenoxyacetic acid (2,4-D) and 8 g/L of Agar, pH=5.8)contains at least one kind of known Agrobacterium-inhibiting antibiotics(cephalosporin) without the selective agent for plant transfectants(Step 3: recovery step). Optionally, the young embryo was cultivated ona solid medium culture containing antibiotics but without selectiveagent so as to eliminate Agrobacterium and to provide a recovery periodfor the infected cells. Then, the inoculated young embryo was cultivatedon a medium containing selective agent (mannose) and the transfected,growing callus was selected (Step 4: selection step). Optionally, theyoung embryo was cultivated on a selective solid medium containingselective agent (4.3 g/L of MS salt, MS vitamins, 300 mg/L of casein, 5g/L of sucrose, 12.5 g/L of mannose, 1 mg/L of 2,4-dichlorophenoxyaceticacid (2,4-D) and 8 g/L of Agar, pH=5.8), resulting the selective growthof the transfected cells. Then, callus regenerated into plants (Step 5:regeneration step). Optionally, the callus was cultivated on a solidmedium containing selective agent (MS differentiation medium and MSrooting medium) to regenerate into plants.

The obtained resistant callus was transferred to said MS differentiationmedium (4.3 g/L MS salt, MS vitamins, 300 mg/L of casein, 30 g/L ofsucrose, 2 mg/L of 6-benzyladenine, 5 g/L of mannose and 8 g/L of Agar,pH=5.8) and cultivated and differentiated at 25° C. The differentiatedseedlings were transferred to said MS rooting medium (2.15 g/L of MSsalt, MS vitamins, 300 mg/L of casein, 30 g/L of sucrose, 1 mg/Lindole-3-acetic acid and 8 g/L of agar, pH=5.8) and cultivated to about10 cm in height at 25° C. Next, the seedlings were transferred to andcultivated in the greenhouse until fructification. In the greenhouse,the maize plants were cultivated at 28° C. for 16 hours and at 20° C.for 8 hours every day.

2. Verification of Transgenic Corn Plants with Inserted 24DT02 GeneUsing TaqMan Technique

100 mg of leaves from every transfected corn plant (corn planttransfected with 24DT02 nucleotide sequence, 24DT02 substitutednucleotide sequence, 24DT02 truncated nucleotide sequence, 24DT02 addednucleotide sequence or control nucleotide sequence, respectively) wastaken as sample respectively. Genomic DNA thereof was extracted usingDNeasy Plant Maxi Kit (Qiagen) and the copy number of 24DT02 gene wasquantified through Taqman probe-based fluorescence quantitative PCRassay. Wild type maize plant was taken as a control and analyzedaccording to the processes as described above. Experiments were carriedout in triplicate and the results were the mean values.

The specific method for detecting the copy number of 24DT02 gene wasdescribed as follows:

Step 11: 100 mg of leaves from every transfected corn plant (corn planttransfected with 24DT02 nucleotide sequence, 24DT02 substitutednucleotide sequence, 24DT02 truncated nucleotide sequence, 24DT02 addednucleotide sequence or control nucleotide sequence, respectively) andwild type corn plant was taken and grinded into homogenate in a mortarin liquid nitrogen respectively. It was in triplicate for each sample.

Step 12: the genomic DNAs of the samples above were extracted usingDNeasy Plant Mini Kit (Qiagen) following the product instructionthereof.

Step 13: the genome DNA concentrations of the above samples weredetermined using NanoDrop 2000 (Thermo Scientific).

Step 14: the genome DNA concentrations were adjusted to the same rangeof 80-100 ng/μl.

Step 15: the copy numbers of the samples were quantified using Taqmanprobe-based fluorescence quantitative PCR assay, the quantified samplewith known copy number was taken as a standard sample and the wild typemaize plant was taken as a control. It was carried out in triplicate forevery sample and the results were the mean values. Primers and theprobes used in the fluorescence quantitative PCR were shown as below.

The following primers and probe were used to detect 24DT02 nucleotidesequence, 24DT02 substituted nucleotide sequence, 24DT02 truncatednucleotide sequence and 24DT02 added nucleotide sequence:

Primer 1:  AGCTGGACGAAGATACATTCTCG; (as shown in SEQ ID NO: 14 in the sequence listing) Primer 2: AGTCCGTCAGGTATTGAGCTGG;  (as shown in SEQ ID NO: 15 in the sequencelisting) Probe 1:  CTGTACCGCGAATGGCTCCAGTATGC; (as shown in SEQ ID NO: 16 in the sequence listing)The following primers and probe were used to detect control sequence:

Primer 3:  TGCGTATTCAATTCAACGACATG; (as shown in SEQ ID NO: 17 in the sequence listing) Primer 4: CTTGGTAGTTCTGGACTGCGAAC;  (as shown in SEQ ID NO: 18 in the sequencelisting) Probe 2:  CAGCGCCTTGACCACAGCTATCCC;(as shown in SEQ ID NO: 19 in the sequence listing)PCR reaction system was as follows:

JumpStart ™ Taq ReadyMix ™ (Sigma) 10 μl  50 × primer/probe mixture 1 μlGenomic DNA 3 μl Water (ddH₂O) 6 μl

Said 50× primer/probe mixture contained 45 μl of each primer (1 mM), 50μl of probe (100 μM) and 860 μl of 1×TE buffer and was stored in anamber tube at 4° C.

PCR reaction conditions were provided as follows:

Step Temperature Time 21 95° C. 5 min 22 95° C. 30 s 23 60° C. 1 min 24back to step 22 and repeated 40 timesData were analyzed using software SDS 2.3 (Applied Biosystems).

The experimental results showed that all the nucleotide sequences of24DT02 nucleotide sequence, 24DT02 substituted nucleotide sequence,24DT02 truncated nucleotide sequence, 24DT02 added nucleotide sequenceand the natural nucleotide sequence have been integrated into thegenomes of the detected corn plants, respectively. Furthermore, all cornplants transfected 24DT02 nucleotide sequence, 24DT02 substitutednucleotide sequence, 24DT02 truncated nucleotide sequence, 24DT02 addednucleotide sequence and the control nucleotide sequence respectivelycontained single copy of 24DT02 gene.

Example 7 Herbicide-resistance Effect Tests of the Transgenic CornPlants

Herbicide resistance effects tests to 2,4-D dimethyl ammonium salt andagroxone of maize plants containing 24DT02 nucleotide sequence, 24DT02substituted nucleotide sequence, 24DT02 truncated nucleotide sequence,24DT02 added nucleotide sequence, control nucleotide sequencerespectively and wild type maize plants (stages V3-V4) were performedrespectively.

Maize plants containing 24DT02 nucleotide sequence, 24DT02 substitutednucleotide sequence, 24DT02 truncated nucleotide sequence, 24DT02 addednucleotide sequence, control nucleotide sequence respectively and wildtype maize plants were taken and spayed with 2,4-D dimethyl ammoniumsalt (8960 g ae/ha, 16-folds concentration in field), agroxone (8960 gae/ha, 16-folds concentration in field) and blank solvent (water)respectively. Prop root development was counted 21 days after spaying.Three strains (S1, S2, and S3) of corn plants transfected with 24DT02nucleotide sequence, two strains (S4 and S5) of corn plants transfectedwith 24DT02 substituted nucleotide sequence, two strains (S6 and S7) ofcorn plants transfected with 24DT02 truncated nucleotide sequence, twostrains (S8 and S9) of corn plants transfected with 24DT02 addednucleotide sequence, two strains (S10 and S11) of corn plantstransfected with control nucleotide sequence and 1 strain of wild type(CK) corn were selected and 10-15 plants from each stain were tested.The results are shown in Table 2.

TABLE 2 Results of herbicide-resistance effect tests of the transgeniccorn T₁ plants Normal Abnormal Ratio of develop- develop- the normallyCorn ment of ment of developed Treatment genotype prop roots prop rootsprop roots Blank solvent S1 15 0 100.00% (water) S2 12 0 100.00% S3 13 0100.00% S4 15 0 100.00% S5 15 0 100.00% S6 14 0 100.00% S7 14 0 100.00%S8 15 0 100.00% S9 13 0 100.00% S10 12 0 100.00% S11 13 0 100.00% CK 160 100.00% 8960 g ae/ha S1 13 3 81.25% 2,4-D dimethyl S2 11 1 91.67%ammonium salt S3 12 2 85.71% (16x 2,4-D) S4 14 2 87.50% S5 15 0 100.00%S6 13 2 86.67% S7 13 1 92.86% S8 12 3 80.00% S9 11 3 78.57% S10 0 10 0%S11 0 11 0% CK 0 16 0% 8960 g ae/ha S1 14 2 87.50% agroxone S2 12 0100.00% (16 x MCPA) S3 12 1 92.31% S4 13 3 81.25% S5 14 1 93.33% S6 14 287.50% S7 13 2 86.67% S8 13 2 86.67% S9 11 2 84.62% S10 0 10 0% S11 0 100% CK 0 16 0%

Results in Table 2 indicate that the 24DT02 gene conferred highresistance against herbicides to the transgenic maize plants, especiallythe phenoxy auxin herbicides (since the monocotyledon plants inherentlyhad certain resistance to phenoxy auxin herbicides, high levels ofresistance appeared); while none of the wild type of corn plants and thecorn plants transfected with control sequences showed high levels ofresistance against herbicides. In addition, resistance levels against2,4-D dimethyl ammonium salt and agroxone of corn plants transformedwith 24DT02 nucleotide sequence, substituted 24DT02 nucleotide sequence,truncated 24DT02 nucleotide sequence and added 24DT02 nucleotidesequence didn't show significant differences.

Both corn and Arabidopsis thaliana plants transfected with 24DT02nucleotide sequence had high herbicide-resistance ability. Some codonsof plant were employed in the herbicide-resistant gene 24DT02 in presentapplication, resulting that the herbicide-resistant gene of presentapplication is suitable to be expressed in plants. 24DT02herbicide-resistant protein of present application has a broadherbicide-resistance spectrum, such as phenoxy auxin herbicides.

Finally what should be explained is that all the above examples aremerely intended to illustrate the technical solutions of presentapplication rather than to restrict present application. Althoughdetailed description of this application has been provided by referringto the examples, one skilled in the art should understand that thetechnical solutions of the application can be modified or equivalentlysubstituted while still fall within the spirit and scope of the presentapplication.

What is claimed is:
 1. An herbicide-resistant protein selected from thegroup consisting of: (a) a protein encoded by a nucleotide sequence setforth in SEQ ID NO: 3; (b) a protein encoded by a nucleotide sequenceset forth in SEQ ID NO: 4; and (c) a protein encoded by a nucleotidesequence set forth in SEQ ID NO:
 5. 2. An herbicide-resistant geneselected from the group consisting of: (a) a nucleotide sequenceencoding the herbicide-resistant protein of claim 1; and (b) anucleotide sequence completely complementary with the nucleotidesequence encoding the herbicide-resistant protein of claim
 1. 3. Amethod for controlling weeds comprising a step of applying an effectiveamount of one or more herbicides to the field planted with plantscomprising the herbicide-resistant gene of claim 2 or a nucleotidesequence encoding SEQ ID NO:
 2. 4. A method for controlling weedscomprising a step of applying an effective amount of one or moreherbicides to the field planted with plants comprising theherbicide-resistant gene of claim 2 or a nucleotide sequence encodingSEQ ID NO: 2 wherein the herbicide resistant gene of claim 2 or thenucleotide sequence encoding SEQ ID NO: 2 is produced from a transgenichost cell selected from the group consisting of plant cells, animalcells, bacteria, yeast, baculovirus, nematodes and algae.
 5. The methodfor controlling weeds of claim 3, wherein the plant is selected from thegroup consisting of soybean, cotton, corn, rice, wheat, beet andsugarcane.
 6. A method for controlling weeds comprising a step ofapplying an effective amount of one or more herbicides to the fieldplanted with plants comprising the herbicide-resistant gene of claim 2or a nucleotide sequence encoding SEQ ID NO: 2 wherein the herbicideresistant gene of claim 2 or the nucleotide sequence encoding SEQ ID NO:2 is co-expressed in the plant with at least a second nucleotidesequence encoding a herbicide-resistant protein different from theherbicide-resistant gene of claim 4 and the nucleotide sequence encodingSEQ ID NO:
 2. 7. The method for controlling weeds of claim 6, whereinthe second nucleotide sequence encodes glyphosate-resistant protein,glufosinate ammonium resistant protein, 4-hydroxyphenylpyruvatedioxygenase, acetolactate synthase, cytochromes protein orprotoporphyrinogen oxidase.
 8. The method for controlling weeds of claim3, wherein the herbicide is a phenoxy auxin.
 9. The herbicide-resistantprotein of claim 1, wherein the nucleotide sequence set forth in SEQ IDNO: 4 is part of a recombinant expression vector.
 10. Theherbicide-resistant protein of claim 1, wherein the nucleotide sequenceset forth in SEQ ID NO: 4 is part of a recombinant expression vector andthe recombinant expression vector comprises a nucleic acid sequence thatis heterologous to SEQ ID NO:
 4. 11. The herbicide-resistant protein ofclaim 1 selected from the group consisting of: (a) a protein encoded bynucleotide sequence set forth in SEQ ID NO: 3; and (b) a protein encodedby nucleotide sequence set forth in SEQ ID NO:
 5. 12. A method forcontrolling weeds comprising a step of applying an effective amount ofone or more herbicides to the field planted with plants comprising theherbicide-resistant gene of claim 2.